Phenanthroindolizidine Alkaloids

ABSTRACT

A group of novel phenanthroindolizidine alkaloid compounds as shown and their use in treating cancer. Also disclosed are methods of using phenanthroindolizidine alkoid compounds in suppressing NO and TNF-α production, inhibiting MEKK1 activity and cyclooxygenase II expression, and treating NO-related disorders.

RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 11/151,600, filed on Jun. 13, 2005, which claims priority to U.S.Provisional Application No. 60/579,033, filed on Jun. 11, 2004. Thecontents of the prior applications are incorporated herein by reference.

BACKGROUND

Nitric oxide (NO) is an important pleiotropic molecule mediating a widerange of physiological and pathophysiological processes. For example, itenhances activity of cyclooxygenase II (COX-II), which is responsiblefor the synthesis of the prostagladins that mediate inflammation, pain,and fever (Liu, Q. et al., Carcinogenesis, 2003, Vol. 24, No.4,637-642.). As another example, it also increases the expression ofsignal kinase MEKK1, which plays a key role in the NF-κB activationpathway (Chou F. P. et al., Toxicology and Applied Pharmacology, 2002,Vol. 181, No. 3: 203-206). Overproduction of NO has been implicated invarious pathological processes, including septic shock, tissue damagefollowing inflammation, cancer, and rheumatoid arthritis.

NO is produced from L-arginine and molecular oxygen by three distinctisoforms of nitric oxide synthase (NOS), i.e., neural NOS (nNOS),endothelial NOS (eNOS), and inducible NOS (iNOS). Among the threeisoforms, iNOS can be induced by endotoxins, cytokines (e.g., TNF-α), ortranscriptional factors (e.g., NF-κB and AP1). Inhibiting expression oractivity of iNOS is a major target for preventing and eliminating NOoverproduction.

SUMMARY

This invention is based on the discovery that certainphenanthroindolizidine alkaloid compounds suppress NO production,inhibit MEKK1 activity, and exert anti-cancer effect.

One aspect of the present invention relates to the compounds of FormulaI:

wherein each of R₁, R₅, R_(6,) R₇, R₈, R₉, R₁₀, R₁₁, R₁₂, R₁₃, R₁₄, R₁₅,and R₁₆, independently, is H, halogen, R, OH, OR, NH₂, NHR, or NRR′; andeach of R₂, R₃, and R_(4,) independently, is halogen, R, OH, OR, NH₂,NHR, or NRR′; each of R and R′, independently, being alkyl, aryl,cyclyl, heteroaryl, or heterocyclyl; X is an anion; and n is theabsolute value of the charge of X.

Referring to Formula I, one subset of the compounds feature that each ofR₉, R₁₀, R₁₁, R₁₂, R₁₃, R₁₄, R₁₅, and R₁₆ is H. Another subset of thecompounds feature that each of R₂, R₃, R_(4,) R₆, and R₇ is OCH₃.

An example of the above-described compounds is shown as Compound 1below.

The above-described compounds possess anti-cancer activity. Thus,another aspect of this invention relates to a method of treating cancerby administering to a subject in need thereof an effective amount of oneof the compounds. The term “cancer” used herein includes all types ofcancerous growths or oncogenic processes, metastatic tissues ormalignantly transformed cells, tissues, or organs, irrespective ofhistopathologic type, or stage of invasiveness. Examples of cancersinclude, but are not limited to, carcinoma and sarcoma such as leukemia,sarcomas, osteosarcoma, lymphomas, melanoma, ovarian cancer, skincancer, testicular cancer, gastric cancer, pancreatic cancer, renalcancer, breast cancer, prostate colorectal cancer, cancer of head andneck, brain cancer, esophageal cancer, bladder cancer, adrenal corticalcancer, lung cancer, bronchus cancer, endometrial cancer, nasopharyngealcancer, cervical or hepatic cancer, and cancer of unknown primary site.

Another aspect of this invention relates to a method for lowering nitricoxide production (via inhibiting expression or activity of iNOS),lowering TNF-α production, lowering COX-II expression, or inhibitingMEKK1 activity. The method includes administering to a subject in needthereof an effective amount of a compound of Formula II:

wherein each of

and

,independently, is a single bond or a double bond; Y is N or N⁺→O⁻, when

is a single bond; or Y is N⁺and a counterion coexists in the compound,when

is a double bond; and each of R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₀,R₁₁, R₁₂, R₁₃, R₁₄, R₁₅ and R₁₆, independently, is H, halogen, R, OH,OR, NH₂, NHR, or NRR′; each of R and R′, independently, being alkyl,aryl, cyclyl, heteroaryl, or heterocyclyl.

Referring to Formula II, one subset of the compounds feature that eachof R₂, R₃, R_(6,) and R₇ is OCH₃ and each of R₁, R_(4,) R₅, and R₈ is H.Another subset of the compounds feature that each of R₁, R₅, and R₈ isH, R₄ is OCH₃, and each of R₉, R₁₀, R₁₁, R₁₂, R₁₃, R₁₄, R₁₅, and R₁₆ isH.

Four exemplary compounds of Formula II are shown below.

As the compounds of Formula II suppress nitric oxide production, theycan be used to treat nitric oxide-related disorders. The nitricoxide-related disorder refers to a disorder associated to overproductionof nitric oxide. Thus, another aspect of this invention relates to amethod for treating a nitric oxide-related disorder. The method includesadministering to a subject in need thereof an effective amount of acompound of Formula III:

wherein each of R₁, R₂, R₃, R_(4,) R₅, R_(6,) R₇, R₈, R₉, R₁₀, R_(11,)R₁₂, R₁₃, R₁₄, R₁₅, and R₁₆, independently, is H, halogen, R, OH, OR,NH₂, NHR, or NRR′; each of R and R′, independently, being alkyl, aryl,cyclyl, heteroaryl, or heterocyclyl; X is an anion; and n is theabsolute value of the charge of X.

Alternatively, the compound used in the just-described method is onehaving Formula IV:

wherein each of R₁, R₅, R_(6,) R₇, R₈, R₉, R₁₀, R₁₁, R₁₂, R₁₃, R₁₄, R₁₅,and R₁₆, independently, is H, halogen, R, OH, OR, NH₂, NHR, or NRR′; R₂,R₃, and R_(4,) independently, is halogen, R, OH, OR, NH₂, NHR, or NRR′;each of R and R′, independently, being alkyl, aryl, cyclyl, heteroaryl,or heterocyclyl.

Referring to Formula III, one subset of the compounds feature that eachof R₉, R₁₀, R₁₁, R₁₂, R₁₃, R₁₄, R₁₅, and R₁₆ is H. Another subset of thecompounds feature that each of R₂, R₃, R₆, and R₇ is OCH₃.

Referring to Formula IV, one subset of the compounds feature that R₉ isOH. Another subset of the compounds feature that each of R₁₀, R₁₁, R₁₂,R₁₃, R₁₄, R₁₅, and R₁₆ is H.

The term “alkyl” refers to a straight or branched hydrocarbon,containing 1-10 carbon atoms. Examples of alkyl groups include, but arenot limited to, methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, andt-butyl.

The term “aryl” refers to a 6-carbon monocyclic, 10-carbon bicyclic,14-carbon tricyclic aromatic ring system wherein each ring may have 1 to4 substituents. Examples of aryl groups include, but are not limited to,phenyl, naphthyl, and anthracenyl.

The term “cyclyl” refers to a saturated and partially unsaturated cyclichydrocarbon group having 3 to 12 carbons, preferably 3 to 8 carbons, andmore preferably 3 to 6 carbons, wherein the cyclyl group may beoptionally substituted. Examples of cyclyl groups include, withoutlimitation, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl,cyclohexyl, cyclohexenyl, cycloheptyl, and cyclooctyl.

The term “heteroaryl” refers to an aromatic 5-8 membered monocyclic,8-12 membered bicyclic, or 11-14 membered tricyclic ring system having1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9heteroatoms if tricyclic, said heteroatoms selected from O, N, or S(e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S ifmonocyclic, bicyclic, or tricyclic, respectively), wherein each ring mayhave 1 to 4 substituents. Examples of heteroaryl groups include pyridyl,furyl or furanyl, imidazolyl, benzimidazolyl, pyrimidinyl, thiophenyl orthienyl, quinolinyl, indolyl, thiazolyl, and the like.

The term “heterocyclyl” refers to a nonaromatic 5-8 membered monocyclic,8-12 membered bicyclic, or 11-14 membered tricyclic ring system having1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9heteroatoms if tricyclic, said heteroatoms selected from O, N, or S(e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S ifmonocyclic, bicyclic, or tricyclic, respectively), wherein 0, 1, 2 or 3atoms of each ring may be substituted by a substituent. Examples ofheterocyclyl groups include piperazinyl, pyrrolidinyl, dioxanyl,morpholinyl, tetrahydrofuranyl, and the like.

Alkyl, aryl, cyclyl, heteroaryl, and heterocyclyl mentioned hereininclude both substituted and unsubstituted moieties. Examples of asubstituent include, but are not limited to, halo, hydroxyl, amino,cyano, nitro, mercapto, alkoxycarbonyl, amido, carboxy, alkanesulfonyl,alkylcarbonyl, carbamido, carbamyl, carboxyl, thioureido, thiocyanato,sulfonamido, alkyl, alkenyl, alkynyl, alkyloxy, aryl, heteroaryl,cyclyl, heterocyclyl, in which alkyl, alkenyl, alkynyl, alkyloxy, aryl,heteroaryl cyclyl, and heterocyclyl are optionally further substitutedwith alkyl, aryl, heteroaryl, halogen, hydroxyl, amino, mercapto, cyano,or nitro.

The term “anion” refers to a negatively charged ion. Examples of ananion include, but are not limited to, Cl⁻, Br⁻, ⁻, SO₄ ²⁻, PO₄ ³⁻, ClO₄⁻, CH₃CO₂ ⁻, and CF₃CO₂ ⁻.

Also within the scope of this invention is a composition containing oneor more of the compounds of Formula I and a pharmaceutically acceptablecarrier for use in treating cancer, as well as the use of such acomposition for the manufacture of a medicament for treating cancer. Inaddition, this invention includes a composition containing one or moreof the compounds of Formula III or Formula IV and a pharmaceuticallyacceptable carrier for use in treating a nitric oxide related disorder,as well as the use of such a composition for the manufacture of amedicament for treating a nitric oxide related disorder.

The details of many embodiments of the invention are set forth in thedescription below. Other features, objects, and advantages of theinvention will be apparent from the description and the claims.

DETAILED DESCRIPTION

Some of the above-described phenanthroindolizidine alkaloid compoundscan be isolated from plant parts. For example, Compound 2, 3, and 4 canbe isolated from leaves of Ficus septica (Wu et al. Heterocycles, 2002,2401). Other compounds can be prepared via simple transformation fromone or more of naturally-occurring compounds. For example, Compound 1can be prepared from Compound 4 via dehydroxylation and dehydration.Scheme 1 shown below depicts the reaction equation of making Compound 1.

The chemicals used in the above-mentioned isolation and synthesis mayinclude, for example, solvents, reagents, catalysts, and protectinggroup and deprotecting group reagents. The synthetic method describedabove may also additionally include steps, either before or after thesteps described specifically herein, to add or remove suitableprotecting groups in order to ultimately allow synthesis of the desiredphenanthroindolizidine alkaloid compounds. Synthetic chemistrytransformations and protecting group methodologies (protection anddeprotection) useful in synthesizing applicable phenanthroindolizidinealkaloid compounds are known in the art and include, for example, thosedescribed in R. Larock, Comprehensive Organic Transformations, VCHPublishers (1989); T. W. Greene and P. G. M. Wuts, Protective Groups inOrganic Synthesis, 3^(rd) Ed., John Wiley and Sons (1999); L. Fieser andM. Fieser, Fieser and Fieser's Reagents for Organic Synthesis, JohnWiley and Sons (1994); and L. Paquette, ed., Encyclopedia of Reagentsfor Organic Synthesis, John Wiley and Sons (1995) and subsequenteditions thereof.

The phenanthroindolizidine alkaloids mentioned above may contain one ormore asymmetric centers. Thus, they occur as racemates and racemicmixtures, single enantiomers, individual diastereomers, diastereomericmixtures, or cis- or trans- isomeric forms. All such isomeric forms arecontemplated.

This invention features a method of inhibiting iNOS promoter activity,suppressing nitric oxide production or inhibiting TNF-α, suppressingCOX-II expression, inhibiting MEKK1 activity by administering to asubject in need thereof an effective amount of a phenanthroindolizidinealkaloid. The term “an effective amount” refers to the amount of thecompound that is required to confer one of the above-described effectson the subject. The effective amount varies, as recognized by thoseskilled in the art, depending on the types of the effects, route ofadministration, excipient usage, and the possibility of co-usage withother treatment.

The phenanthroindolizidine alkaloid compounds of Formula I possessanti-cancer activity and the phenanthroindolizidine alkaloid compoundsof Formula III or IV inhibit NO production. Thus, this invention coversa method of treating cancer or a nitric oxide-related disorder byadministering a phenanthroindolizidine alkaloid compound. Also withinthe scope of this invention is a pharmaceutical composition containingan effective amount of the phenanthroindolizidine alkaloid compound anda pharmaceutical acceptable carrier. The term “treating” refers toadministering the compound to a subject who has a disorder, (i.e.,cancer or a nitric oxide-related disorder), or has a symptom of thedisorder, or has a predisposition toward the disorder, with the purposeto cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve,or affect the disorder, the symptoms of the disorder, or thepredisposition toward the disorder.

A nitric oxide-related disorder is a disorder or condition associated tooverproduction of NO, such as an inflammatory disease. An inflammatorydisease is characterized by a local or systemic, acute or chronicinflammation. Examples of an inflammatory diseases include systemiclupus erythematosus, encephalitis, meningitis, arthritis,atherosclerosis, hepatitis, sepsis, sarcoidosis, psoriasis, Type Idiabetes conjunctivitis, asthma, arteriosclerosis, chronic obstructivepulmonary disease, sinusitis, dermatitis, inflammatory bowel disease,ulcerative colitis, Crohn's disease, Behcet's syndrome, and graftrejection.

To practice the method of the present invention, a composition havingone or more of the above-described phenanthroindolizidine alkaloidcompounds can be administered parenterally, orally, nasally, rectally,topically, or buccally. The term “parenteral” as used herein refers tosubcutaneous, intracutaneous, intravenous, intramuscular,intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal,intralesional, or intracranial injection, as well as any suitableinfusion technique.

A sterile injectable composition can be a solution or suspension in anon-toxic parenterally acceptable diluent or solvent, such as a solutionin 1,3-butanediol. Among the acceptable vehicles and solvents that canbe employed are mannitol and water. In addition, fixed oils areconventionally employed as a solvent or suspending medium (e.g.,synthetic mono- or diglycerides). Fatty acid, such as oleic acid and itsglyceride derivatives are useful in the preparation of injectables, asare natural pharmaceutically acceptable oils, such as olive oil orcastor oil, especially in their polyoxyethylated versions. These oilsolutions or suspensions can also contain a long chain alcohol diluentor dispersant, carboxymethyl cellulose, or similar dispersing agents.Other commonly used surfactants such as Tweens or Spans or other similaremulsifying agents or bioavailability enhancers which are commonly usedin the manufacture of pharmaceutically acceptable solid, liquid, orother dosage forms can also be used for the purpose of formulation.

A composition for oral administration can be any orally acceptabledosage form including capsules, tablets, emulsions and aqueoussuspensions, dispersions, and solutions. In the case of tablets,commonly used carriers include lactose and corn starch. Lubricatingagents, such as magnesium stearate, are also typically added. For oraladministration in a capsule form, useful diluents include lactose anddried corn starch. When aqueous suspensions or emulsions areadministered orally, the active ingredient can be suspended or dissolvedin an oily phase combined with emulsifying or suspending agents. Ifdesired, certain sweetening, flavoring, or coloring agents can be added.

A nasal aerosol or inhalation composition can be prepared according totechniques well known in the art of pharmaceutical formulation. Forexample, such a composition can be prepared as a solution in saline,employing benzyl alcohol or other suitable preservatives, absorptionpromoters to enhance bioavailability, fluorocarbons, and/or othersolubilizing or dispersing agents known in the art. A composition havingone or more active phenanthroindolizidine alkaloid compounds can also beadministered in the form of suppositories for rectal administration.

The carrier in the pharmaceutical composition must be “acceptable” inthe sense that it is compatible with the active ingredient of thecomposition (and preferably, capable of stabilizing the activeingredient) and not deleterious to the subject to be treated. One ormore solubilizing agents can be utilized as pharmaceutical excipientsfor delivery of an active phenanthroindolizidine alkaloid compound.Examples of other carriers include colloidal silicon oxide, magnesiumstearate, cellulose, sodium lauryl sulfate, and D&C Yellow # 10.

The above-described effects of a phenanthroindolizidine alkaloidcompound can be tested by an in vitro or in vivo assay. For example,compounds of Formula I can be preliminarily screened by in vitro assaysin which the compounds are tested for their efficacy in inhibitingcancer cell growth. Compounds that demonstrate high efficacy in thepreliminary screening can be further evaluated by in vivo methods wellknown in the art to evaluate their activity in treating cancer.

Similarly, compounds of Formula III or IV can be screened by an in vitroassay for their activity of suppressing nitric oxide production.Compounds that demonstrate high activity can be further evaluated by invivo assays to evaluate their activity of treating a nitricoxide-related disorder.

The specific examples below are to be construed as merely illustrative,and not limitative of the remainder of the disclosure in any waywhatsoever. Without further elaboration, it is believed that one skilledin the art can, based on the description herein, utilize the presentinvention to its fullest extent. All publications cited herein arehereby incorporated by reference in their entirety.

EXAMPLE 1 Preparation of Compounds 1-4

Compounds 2, 3, and 4 were isolated from the leaves of Ficus septicaaccording to the method described in Wu et al. Heterocycles, 2002, 2401.Compound 4 was dehydroxylated and dehydrated to afford Compound 1.

EXAMPLE 2 Biological Assays

Inhibition of iNOS promoter activity:

RAW 264.7 cells (or A549 cells) were seeded in 24-well plates (9×10⁴cells/well) and grown overnight in sodium pyruvate-free DMEM with 4 mMglutamine containing 1% non-essential amino acids (BiologicalIndustries, Israel) with antibiotics and 10% heat-inactivated fetalbovine serum or 10% fetal calf serum (Biological Industries, Israel).The cells were then co-transfected with murine or human iNOS or murineCOX-II promoter-luciferase reporter plasmids (100 ng/well) andpCMV-β-gal (100 ng/well) using FuGene6 (Roche, Germany) following theprotocol provided by the manufacturer. Murine iNOS promoter luciferaseplasmids, human iNOS promoter-luciferase reporter plasmids (pGL3-8296),and murine COX-II promoter-luciferase reporter plasmids were generouslyprovided by Drs. Charles J. Lowenstein (John Hopkines University), JoelMoss (National Institute of Health), and Yu-Chih Liang (Taipei MedicalUniversity), respectively.

The cells were incubated for 24 h and the culture medium was replacedwith one containing 5 μg/ml lipopolysaccharide (LPS), 20 ng/ml IFNγ, andeach of Compounds 1-4 (final concentration: 10 μM). After 18-20 h, themedium was removed and 150 μl of Glo lysis buffer was added to eachwell. The resultant lysates were subjected to the luciferase andβ-galactosidase assays. Transfection efficiency was normalized byβ-galactosidase activity.

The luciferase and β-galactosidase assays were performed using aSTEADY-GLO luciferase assay system (Promega) and Galacto-Star assaysystem (Tropix) according to the manufacturer's instructions.Luminescence was measured in a TOPCOUNT.NXT™ Microplate Scintillationand Luminescence Counter (Packard Inc.). The results were normalized toβ-galactosidase activity derived from co-transfected LacZ gene under thecontrol of a constitutive promoter.

The results show that Compounds 2 and 4 effectively inhibited iNOSpromoter activity.

Inhibition of AP1 and NF-κB activity:

RAW 264.7 cells (or A549 cells) were seeded in 24-well plates (10⁶cells/well) 4-6 h before they were transfected with pNF-κB-Luc(Stratagene Corp., Calif.) (100 ng/well) and pCMV-β-gal (100 ng/well),or pAP 1-Luc (Stratagene Corp., Calif.) (200 ng/well) alone. Thetransfected cells were incubated for 24 h and then washed with theculture medium twice. After 18 h of incubation, the cells were treatedwith LPS (10 μg/ml)/IFNγ (20 ng/ml) and Compound 2 (final concentration:0.3, 1, 3, or 10 μM) for 5 h. The cells were subjected to the luciferaseand β-galactosidase assays. Of note, in the assay involving pNF-κB-Luc,the culture medium was free of serum; and in the assay involving pAP1-Luc, the culture medium did not include non-essential amino acids. Theresults show that Compound 2 at the concentration higher than 1 μMinhibited AP 1 activity, but not NF-κB activity.

In a similar manner, RAW264.7 cells were transfected with pNF-κB-Lucplasmids (100 ng/well) or pAP 1-Luc (100 ng/well) as well as pFC-MEKK(Stratagene Corp., Calif.) (50 ng/well) and/or pCMV-β-gal plasmids (100ng/well) and the transfected cells were subjected to the luciferase andβ-galactosidase assays. The results show that Compound 2 at theconcentration of 3 μM or higher inhibited activity of AP 1 or pNF-κB inthe cells which was treated with LPS/IFNγ and had over-expression ofMEKK1. The results suggest that Compound 2 inhibited MEKK1 activity orexpression. The extent of the inhibition was comparable to that observedin the cells which had over-expression of MEKK1, but was not treatedwith LPS/IFNγ.

In addition, it was observed that Compound 2 enhanced phosphorylation ofAkt (a serine/threonine kinase) and ATF-2 (a transcriptional factor),and inhibited expression of c-Jun (a component of the transcriptionfactor AP1) in LPS/IFNγ-treated RAW264.7 cells. Also, Compound 2 andanother P13K/Akt inhibitor, e.g., LY294002 (Vlahos, C. J. et al. J.Immunol. 1995, 154: 2413), significantly inhibited NF-κB activity.

Inhibition of carcinoma cell growth:

Two cancer cell lines, HONE-1 (nasopharyngeal carcinoma) and NUGC-3(gastric cancer), were maintained in DMEM medium containing 10% fetalbovine serum, seeded in 96-well plates (4500 and 6000 cells/well,respectively), and incubated under CO₂ at 37° C. for 24 h. The cellswere treated with at least five different concentrations of testcompounds in a CO₂ incubator for 72 h. The number of viable cells wasestimated using the tetrazolium dye reduction assay (MTS assay)according to the protocol provided by the manufacturer (Promega,Madison, Wis., USA). The absorbance was measured at 490 nm on a Wallac1420 VICTOR2 Multilabel counter (Perkin-Elmer, Boston, Mass.). Theresults of these assays were used to obtain the dose-response curvesfrom which IC₅₀ (nM) values were determined (an IC₅₀ value representsthe concentration (nM) of the test compound at which it produces a 50%cell growth inhibition after 3 days of incubation). The results showthat Compound 1, 2, and 4 inhibited proliferation of HONE-1 and NUGC-3cells.

Inhibition of nitric oxide and TNF α production and iNOS and COX-IIexpression:

RAW 264.7 cells were seeded (70,000 cells/well) and cultured in 96-wellculture plate. After 24 h incubation, the medium was replaced with onecontaining LPS (5 ug/ml)/IFNγ (20 ng/ml) and each of Compounds 1-4(final concentration: 3, 5, or 6 μM) was added at differentconcentrations. After 18-24 h, the supernatants were subjected to themeasurement of nitric oxide production using the Nitrate/Nitrite assaykit (Cayman Chemical). Nitric oxide levels were measured as theaccumulation of nitrite and nitrate in the incubation medium. Nitratewas reduced to nitrite with nitrate reductase and determinedspectrophotometrically with the Griess reagent at OD₄₀₅. The attachedcells were subjected to cytotoxicity measurement using the MTS assay.The results show that Compounds 1-4 inhibited production of NO.

An ELISA kit (R & D Systems Inc., USA) was used to measure TNF α proteinin cell culture supernatants using according to the manufacturer'sinstructions. The results show that Compound 2 inhibited production ofTNF α.

Levels of iNOS, COX-II, and β-actin (control) were measured byimmunoblotting with anti-iNOS antibody (Biomol), anti-COX-II antibody(Upstate), and anti-β-actin antibody (Chemicon), respectively. The celllysates were subjected to SDS-PAGE and the separated proteins wereelectrophoretically transferred to nitrocellulose membranes. Themembranes were incubated, respectively, with blocking solution for 1 h,primary antibody for 2 h, and secondary antibody for 1 h.Antigen-antibody complexes were detected using ECL detection reagents(Perkin Elmer, Western Blot Chemiluminescence Reagent Plus) according tothe manufacturer's instructions. The results show that Compound 2lowered both iNOS and COX-II levels.

OTHER EMBODIMENTS

All of the features disclosed in this specification may be combined inany combination. Each feature disclosed in this specification may bereplaced by an alternative feature serving the same, equivalent, orsimilar purpose. Thus, unless expressly stated otherwise, each featuredisclosed is only an example of a generic series of equivalent orsimilar features.

From the above description, one skilled in the art can easily ascertainthe essential characteristics of the present invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions. For example, compounds structurally analogous toabove-described phenanthroindolizidine alkaloid compounds also can bemade, screened for the above-described activities and used to practicethis invention. Thus, other embodiments are also within the claims.

1. A method for treating cancer, comprising administering to a subjectin need thereof an effective amount of a compound of the followingformula:

wherein each of R₁, R₅, R₆, R₇, R₈, R₉, R₁₀, R₁₁, R₁₂, R₁₃, R₁₄, R₁₅,and R₁₆, independently, is H, halogen, R, OH, OR, NH₂, NHR, or NRR′; andeach of R₂, R₃, and R₄, independently, is halogen, R, OH, OR, NH₂, NHR,or NRR′; each of R and R′, independently, being alkyl, aryl, cyclyl,heteroaryl, or heterocyclyl; X is an anion; and n equals the absolutevalue of the charge of X so that the overall charge of the compound is0.
 2. The method of claim 1, wherein each of R₉, R₁₀, R₁₁, R₁₂, R₁₃,R₁₄, R₁₅, and R₁₆ is H.
 3. The method of claim 2, wherein each of R₆ andR₇ is OCH₃.
 4. The method of claim 3, wherein each of R₁, R₅, and R₈ isH.
 5. The method of claim 4, wherein each of R₂, R₃, and R₄ is OCH₃. 6.The method of claim 1, wherein each of R₂, R₃, R₄, R₆, and R₇ is OCH₃.7. The method of claim 6, wherein each of R₁, R₄, R₅, and R₈ is H.
 8. Amethod for treating a nitric oxide-related disorder, comprisingadministering to a subject in need thereof an effective amount of acompound of the following formula:

wherein each of R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₀, R₁₁, R₁₂, R₁₃,R₁₄, R₁₅, and R₁₆, independently, is H, halogen, R, OH, OR, NH₂, NHR, orNRR′; each of R and R′, independently, being alkyl, aryl, cyclyl,heteroaryl, or heterocyclyl; X is an anion; and n equals the absolutevalue of the charge of X so that the overall charge of the compound is0.
 9. The method of claim 8, wherein each of R₉, R₁₀, R₁₁, R₁₂, R₁₃,R₁₄, R₁₅, and R₁₆ is H.
 10. The method of claim 9, wherein each of R₂,R₃, R₆, and R₇ is OCH₃.
 11. The method of claim 8, wherein each of R₂,R₃, R₆, and R₇ is OCH₃.
 12. The method of claim 8, wherein the nitricoxide-related disorder is an inflammatory disease.
 13. The method ofclaim 12, where in the inflammatory disease is arthritis oratherosclerosis.
 14. A method for treating a nitric oxide-relateddisorder, comprising administering to a subject in need thereof aneffective amount of a compound of the following formula:

wherein each of R₁, R₅, R₆, R₇, R₈, R₉, R₁₀, R₁₁, R₁₂, R₁₃, R₁₄, R₁₅,and R₁₆, independently, is H, halogen, R, OH, OR, NH₂, NHR, or NRR′; R₂,R₃, and R₄, independently, is halogen, R, OH, OR, NH₂, NHR, or NRR′;each of R and R′, independently, being alkyl, aryl, cyclyl, heteroaryl,or heterocyclyl.
 15. The method of claim 14, wherein each of R₉ is OH.16. The method of claim 15, wherein R₂, R₃, R₄, R_(6,) and R₇ is OCH₃.17. The method of claim 16, wherein each of R₁, R₅, and R₈ is H.
 18. Themethod of claim 14, wherein the nitric oxide-related disorder is aninflammatory disease.
 19. The method of claim 18, where in theinflammatory disease is arthritis or atherosclerosis.
 20. A method forsuppressing nitric oxide production, comprising administering to asubject in need thereof an effective amount of a compound of thefollowing formula:

wherein each of

and

, independently, is a single bond or a double bond; Y is N or N⁺→O⁻,when

is a single bond; or Y is N⁺ and a counterion coexists in the compound,when

is a double bond; and each of R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₀,R₁₁, R₁₂, R₁₃, R₁₄, R₁₅ and R₁₆, independently, is H, halogen, R, OH,OR, NH₂, NHR, or NRR′; each of R and R′, independently, being alkyl,aryl, cyclyl, heteroaryl, or heterocyclyl.
 21. The method of claim 20,wherein each of R₂, R₃, R₆, and R₇ is OCH₃.
 22. The method of claim 21,wherein each of R₁, R₄, R₅, and R₈ is H.
 23. The method of claim 22,wherein each of

and

is a single bond, and Y is N.